SUMMARY A major goal of bacterial cell biology is to understand the mechanisms underlying the assembly and growth of the cell envelope. In addition to addressing a fundamental biological question, studies in this area have significant consequences for human health. The envelope serves as both a major target for antibiotics and, in the case of gram-negative bacteria, a formidable barrier that prevents drugs from reaching their target. Thus, understanding of the mechanisms required for construction of the gram-negative envelope will help identify new vulnerabilities in the process to target for antibiotic development. The peptidoglycan (PG) cell wall layer of the envelope is critical for cell shape and integrity. It is composed of long glycans connected by crosslinks between attached peptides to form a net-like structure that surrounds and protects the cytoplasmic membrane from osmotic lysis. In E. coli and many other bacilli, the processes of cell elongation and cell division are carried out by multi-protein cell wall synthetic machines called the Rod system and the divisome, respectively. The Rod system is organized by filaments of the actin-like protein MreB whereas the tubulin-like protein FtsZ governs cell division. Despite years of study, the function of proteins within these machineries have remained surprisingly ill-defined. Until recently, it has even been unclear which enzymes synthesize PG within these complexes. Because they were the only factors known to possess PG glycan polymerase activity, the class A penicillin-binding proteins (aPBPs) have traditionally been thought to fill this role. However, we changed this view by demonstrating that SEDS (shape, elongation, division, and sporulation) proteins in the Rod system (RodA) and divisome (FtsW) have PG polymerase activity and work in conjunction with PG crosslinking enzymes called class B PBPs (bPBPs) to build the cell wall. Our findings have therefore led us to propose a new model for cell wall synthesis where SEDS-bPBP complexes form the core PG synthases of cytoskeletally organized machineries, with RodA-PBP2 and FtsW-PBP3 comprising the Rod system and divisome synthases, respectively. The experiments described in this proposal will build on our recent breakthrough by taking advantage of a newly developed genetic system for the isolation of mutants encoding inactive or hyperactive Rod systems. Several mutants isolated provide a foundation for defining how RodA polymerase activity is regulated within the Rod system and coupled with the crosslinking activity of PBP2. Additional genetic analyses will also be initiated aimed at defining the function of other conserved yet poorly characterized components of the Rod system and their potential role in regulating the activity of the core RodA-PBP2 synthase. Finally, biochemical and genetic studies will be initiated to understand how the related FtsW-PBP3 synthase is regulated within the divisome. Overall, the results will significantly advance our understanding of cell wall biogenesis by multi-protein PG synthetic machineries, and the knowledge gained will aid the discovery of new classes of antibacterial agents that target these systems.